Facioscapulohumeral muscular dystrophy (FSHD) is a progressively debilitating syndrome that is the nation's third most common type of dystrophy, yet the development of directed therapies remains hampered by our poor understanding of its molecular basis. Recent progress using a mouse model system suggests that overexpression of the FRG1 gene is sufficient to cause a number of FSHD symptoms. Thus a promising avenue for therapeutic intervention is the reduction of FRG1 expression and/or function. Several lines of evidence suggest that FRG1 associates with the RNA splicing machinery and may alter normal splicing when overexpressed, but the specific biochemical activity of FRG1 remains unknown. In this project we will utilize the powerful genetic approaches available in the fruitfly Drosophila melanogaster to identify molecular pathways through which FRG1 functions. Using the GAL4-UAS system we will target overexpression of FRG1 specifically to the indirect flight muscle of the adult fly. The morphology and function of this muscle are easily assessed and it therefore an ideal target for genetic studies. Because FSHD has a variety of extramuscular effects we will also examine the effects of FRG1 overexpression on the Drosophila compound eye, a highly ordered tissue that is frequently exploited for analysis of gene function. Using overexpression phenotypes in these tissues we will carry out genetic screens to identify modifier mutations that reverse the harmful effects of excess FRG1 expression. Because such screens are carried out in unbiased manner they offer the potential to identify novel interacting factors that would not otherwise have been considered or investigated. As no specific biochemical activity has previously been linked with FSHD, these factors will provide new targets for strategies aimed at therapeutic inhibition of FRG1 function. In parallel studies we will exploit the extensive knowledge of RNA splicing mechanisms already developed in Drosophila to ask whether FRG1 is a required component of the spliceosome. Using an in vitro splicing system we will identify the specific steps in splicing and splicing complex assembly and function where FRG1 acts. These studies will thus provide the first genetic and biochemical analysis of FRG1 in Drosophila. By developing Drosophila as a model for FSHD this work will form a basis for future studies that will elaborate a detailed understanding of both the normal molecular function of FRG1 and the effects of its overexpression in muscle. These studies thus offer the potential to uncover novel biochemical pathways that affect muscle function and disease. [unreadable] [unreadable] [unreadable]